Herpes Simplex Virus (HSV) is a well-studied virus. Both distinguishable serotypes of Herpes Simplex Virus (HSV-1 and HSV-2) cause infection and disease ranging from relatively minor fever blisters on lips to severe genital infections, and generalized infections on newborns. HSV-1 and HSV-2 are 50% homologous at the DNA level, and polyclonal antibodies and MAbs to shared epitopes for one are cross-reactive to the other.
HSV-1 and HSV-2 have RR1 proteins (respectively designated ICP6 and ICP10) that contain a unique amino terminal domain. The HSV-2 unique domain codes for a ser/thr-specific PK which has auto- and transphosphorylating activity and has a transmembrane domain. Sequences which code for the PK domain cause neoplastic transformation and are associated with cervical cancer (HSV-2 oncogene). The unique terminal domain of the HSV-1 RR1 protein (ICP6) also has PK activity but it is different from that of the HSV-2 oncogene both structurally and functionally.
Original studies, using enzymatic assay conditions similar to those employed for ICP10 PK, concluded that ICP6 does not have PK activity, although the unique domain is retained (Chung et al., J. Virol. 63:3389-3398, 1989). This was not unexpected since the sequence of the unique PK domains showed only 38% homology (Nikas et al., Proteins:Structure, function and genetics 1:376-384, 1986). Further studies indicated that ICP6 has PK activity but only under different conditions. Also there is controversy as to whether the activity is both auto- and transphosphorylating (see Peng et al., Virology 216:184-196, 1996 for a review of the problem; particularly Table 1). The reason for the different PK activities of the ICP6 and ICP10 proteins is likely to be that the ICP6 PK has its ATP binding sites located distantly from the rest of the catalytic motifs (Cooper et al., J. Virol. 69:4979-4985, 1995). ICP6 also does not have a TM domain and it does not localize to the cell surface (Conner et al., Virology 213:615, 1995). The PK activity of native ICP6 is very weak even under ideal conditions, such that its K.sub.m is 10-fold higher than that of ICP10 PK (Peng et al., Virology 216:184, 1996; Lee and Aurelian, in preparation).
The transforming activity of ICP6 is located within a genome fragment that is distant from that at which the HSV2 oncogene is located. Transformation in this system is only based on focus formation.
It has previously been shown that HSV-2 protein kinase activity is localized at amino acids 1-446 (Chung et al., J. Virol. 63:3389-3398, 1989). Cells which express a protein consisting of ICP10 amino acids 1-446 evidence anchorage independent growth and neoplastic growth. Therefore, the HSV-2 oncogene is located at the DNA sequence encoding ICP10 (SEQ ID NO:2) amino acids 1-446 (Smith et al., Virology 200:598-612, 1994).
Protein kinase (PK) activity is required for neoplastic transformation (Smith et al., Virology 200:598-612, 1994). Transformation is seen in both rodent and human cells (Jariwalla et al., PNAS 77:2279-2283, 1980; Hayashi et al., PNAS 82:8493-8497, 1985; Smith et al., Virology 200:598-612, 1994). Studies therefore have demonstrated that the HSV-2 oncoprotein is located at ICP10 amino acids 1-446.
The minimal ICP10 size required for PK activity is at amino acids 1-283 (pp29.sup.la1) (Luo et al., J. Biol. Chem. 266: 20976-20983, 1991). The PK activity of pp29.sup.la1 has some properties different from the authentic ICP10 PK (Luo et al., J. Biol. Chem. 266: 20976-20983, 1991). The minimal size of the ICP10 transforming protein (HSV-2 oncogene) is at amino acids 1-446. It has been shown that the PK domain encompasses eight catalytic motifs and SH3-binding sites which are involved in interaction with signaling proteins and is located at ICP10 amino acids 1-446 (Chung et al., J. Virol. 63:3389-3398, 1989; Nelson et al., J. Biol. Chem. 271:17021-17027, 1996).
It has also previously been shown that the HSV-2 oncoprotein has intrinsic PK activity. This was shown by demonstrating that ICP10 PK activity is lost through site-directed mutagenesis. The oncogene also has SH3-binding domains at positions 140, 149 and 396, which are required for interaction with signaling proteins. This interaction is required for transforming activity. Site directed mutagenesis was used to identify amino acids required for kinase activity and interaction with signaling proteins. Mutation of Lys.sup.176 or Lys.sup.259 reduced PK activity (5-8 fold) and binding of the .sup.14 C-labeled ATP analog p-fluorosulfonylbenzoyl 5'-adenosine (FSBA), but did not abrogate them. Enzymatic activity and FSBA binding were abrogated by mutation of both Lys residues, suggesting that either one can bind ATP. Mutation of Glu.sup.209 (PK catalytic motif III) virtually abrogated kinase activity in the presence of Mg.sup.2+ or Mn.sup.2+ ions, suggesting that Glu.sup.209 functions in ion-dependent PK activity. ICP10 bound the adaptor protein Grb.sub.2 in vitro. Mutation of the ICP10 proline-rich motifs at position 396 and 149 reduced Grb.sub.2 binding 20- and 2-fold respectively. Binding was abrogated by mutation of both motifs. Grb.sub.2 binding to wild type ICP10 was competed by a peptide for the Grb.sub.2 C-terminal SH3 motif indicating that it involves the Grb.sub.2 C-terminal SH3 (Nelson et al., J. Biol. Chem. 271:17021-17027, 1996).
The construction of the ICP10 PK virus is described by Peng et al. (Virology 216, 184-196, 1996) and Smith et al., (submitted). Briefly, the wild type sequences in a plasmid (TP101) that contains the HSV-2 BamHI E and T fragments were replaced with the 1.8 kb SalI/BglII fragment from pJHL9 [ICP10 mutant deleted in the PK catalytic domain (Luo and Aurelian, J. Biol. Chem. 267:9645-9653, 1992)]. The resulting plasmid, TP9, contains sequences which code for ICP10 deleted in the PK catalytic domain flanked by 4 and 2.8 kb of HSV-2 DNA sequences at the 5' and 3' ends, respectively. The 10 kb HindIII/EcoR1 fragment from TP9 was introduced by marker transfer into a virus (ICP10 RR) in which the RR domain of ICP10 had been replaced with the LacZ gene. The resulting recombinant virus, designated ICP10.DELTA.PK, was obtained by selecting white plaques on a background of blue plaques after staining with X-gal. A few white plaques were picked, purified, and grown in Vero cells with 10% serum (exponentially growing).
There are several known HSV-2 vaccines in the prior art. U.S. Pat. Nos. 4,347,127; 4,452,734; 5,219,567; and 5,171,568 each teach subunit vaccines which provide some protection against HSV-2 infection. These vaccines are inferior to one in which a live, attenuated virus is used. The immunity induced by a subunit vaccine is restricted to the particular protein represented by the subunit, which may not have sufficient protective potential, Additionally it is non-replicating and there is therefore no amplification of the protein which would further reduce immunogenicity. These problems occur in any sub-unit vaccine regardless of whether the method of preparation is via a recombinant protein or a purification of antigen from a virus.
A cross recombinant vaccine, such as disclosed in U.S. Pat. No. 4,554,159, does not suffer from the problems of the subunit vaccines, but contains the oncogene present in HSV-2. Unless care is taken to define and delete the oncogene, the cross recombinant vaccine would induce cancer in the vaccinee.
The cross recombinant of '159 is temperature sensitive. Avirulence may be obtained by selecting temperature resistance, but the temperature of the mouse is 39.degree. C., while that of a man is 37.degree. C. This temperature sensitivity could well render such a cross problematic in a vaccine. A superior method of selection of avirulence is by the removal of genes coding for virulence without respect to the temperature at which the virus replicates. Also, the use of prototypical crosses would preclude the use of gene deleted or inserted mutants.
Due to the many type-common epitopes on HSV-1 and HSV-2, the antibodies in human serum are cross-reactive (Aurelian, L., Royston, I., and Davis, H. J. Antibody to genital herpes simplex virus: Association with cervical atypia and carcinoma in situ. J. Natl. Cancer Inst. 45:455-464, 1970.) It has also been previously shown that cell-mediated immunity cross-reacts (Jacobs, R. P., Aurelian, L., and Cole, G. A. Cell-mediated immune response to herpes simplex virus: Type specific lymphoproliferative responses in lymph nodes draining the site of primary infection. J. Immunol. 116:1520-1525, 1976).
A live vaccine is superior to a dead vaccine because the live vaccine induces herd immunity; it induces different types of immunity, such as mucosal, cell mediated and humoral immmunity; a higher level of immunity is normally obtained because the virus titers are increased through replication within the vaccinee; and finally a live vaccine is of longer duration, thus obviating boosters and lowering initial dosage. HSV-1 is not as desirable as a candidate for a vaccine against herpes because the major clinical problem is the sexually transmitted HSV-2 which is associated with cancer induction. HSV-1 has a 50% homology with HSV-2, and this may lower the response rate against the heterologous strain in the vaccinated population. All known vaccines for HSV-1 or HSV-2 are cross-reactive and provide complete immunity to the other type. Known vaccines are not type specific. However, an absolute necessity for a live herpes vaccine is the removal of the gene responsible for the association with cancer induction, as in the present invention.
Another absolute requirement for a live vaccine is the absence of lesions upon immunization. A desirable trait in the live vaccine would be its ability to cause a reduction in the frequency of recurrent lesions in a person already infected. There is a substantial population already infected with HSV who may have intercourse with uninfected individuals who would benefit from such a vaccine.
The present invention solves all the problems recited above by providing a whole live attenuated HSV-2 in which the HSV-2 has a deletion of the oncogene, and is formulated in a vaccine composition. The present invention provides a method of immunizing a subject against HSV-1 or HSV-2 with said vaccine composition, providing a superior method of conferring immunity upon the subject.